Potentiation of developmentally regulated plant defense response by AtWRKY18, a pathogen-induced Arabidopsis transcription factor

Abstract

AtWRKY18 is a pathogen- and salicylic acid-induced Arabidopsis transcription factor containing the plant-specific WRKY zinc finger DNA-binding motif. In the present study, we have transformed Arabidopsis plants with AtWRKY18 under control of the cauliflower mosaic virus 35S promoter. Surprisingly, transgenic plants expressing high levels of AtWRKY18 were stunted in growth. When expressed at moderate levels, AtWRKY18 potentiated developmentally regulated defense responses in transgenic plants without causing substantial negative effects on plant growth. As they grew from seedling to mature stages, transgenic AtWRKY18 plant showed marked increase in the expression of pathogenesis-related genes and resistance to the bacterial pathogen Pseudomonas syringae, whereas wild-type plants exhibited little enhancement in these defense responses. Potentiation of developmentally regulated defense responses by AtWRKY18 was not associated with enhanced biosynthesis of salicylic acid but required the disease resistance regulatory protein NPR1/NIM1. Thus, AtWRKY18 can positively modulate defense-related gene expression and disease resistance. To study the regulated expression of AtWRKY18, we have identified a cluster of WRKY binding sites in the promoter of the gene and demonstrated that they acted as negative regulatory elements for the inducible expression of AtWRKY18. These negative cis-acting elements may prevent overexpression of AtWRKY18 during the activation of plant defense responses that could be detrimental to plant growth as inferred from the transgenic plants ectopically expressing the transgene.

Figure 1

Overexpression constructs of AtWRKY18. A, Schematic diagrams of the construct 35S-W18 (the full-length AtWRKY18 cDNA sequence placed between the cauliflower mosaic virus 35 promoter and 35S terminator) and the construct 35S-W18Δ (the 3′-untranslated sequence of AtWRKY18 deleted). B, Sequence of the AtWRKY18 3′-untranslated region. The four direct repeats are highlighted (underlined).

Figure 2

Expression of AtWRKY18 in transgenic plants. A, Total RNA was isolated from 4-week-old wild-type plants (Wt) with (+) or without (−) SA treatment (2 mm for 3 h) and untreated T₃ progeny of transgenic lines harboring the 35S-W18 construct, separated on a 1.2% (w/v) agarose-formaldehyde gel, and probed with an AtWRKY18 cDNA fragment. The ethidium bromide stain of rRNA is shown for each lane to allow assessment of equal loading. B, Total RNA was isolated from 4-week-old wild-type plants (Wt) with (+) or without (−) SA treatment (2 mm for 3 h) and untreated T₃ progeny of transgenic lines harboring the 35S-W18Δ construct, separated on a 1.2% (w/v) agarose-formaldehyde gel, and probed with an AtWRKY18 cDNA fragment. The ethidium bromide stain of rRNA is shown for each lane to allow assessment of equal loading. C, Total RNA was isolated from 4-week-old wild-type plants (Wt) with (+) or without (−) SA treatment (2 mm for 3 h) and untreated T₃ progeny of two transgenic lines. Reverse transcription and PCR were performed as described in “Materials and Methods.”

Figure 3

Growth of transgenic lines in soil. Wild-type plants, T₃ progeny of transgenic AtWRKY18Δ line 1 (W18Δ-1), and transgenic AtWRKY18 lines 4 and 5 (W18-4 and W18-5) were germinated and grown in a growth chamber. The plants were photographed 32 d after germination.

Figure 4

Potentiation of PR gene expression in transgenic plants. Total RNA was isolated at indicated times after germination from wild type (Wt) and T₃ progeny of two transgenic lines harboring the 35S-W18 or 35-W18Δ construct, separated on a 1.2% (w/v) agarose-formaldehyde gel, and probed with a PR-1 fragment. The blot was subsequently stripped and reprobed with PR-2 and PR-5. The ethidium bromide stain of rRNA is shown for each lane to allow assessment of equal loading.

Figure 5

Enhanced disease resistance in transgenic plants. Wild type (Wt) and T₃ progeny of transgenic line W18Δ-1 and W18-5 at 3.5-week (A) and 5.5-week (B) ages were treated with water or SA (2 mm). Three days after the treatment, the plants were inoculated with P. syringae DC3000 (OD₆₀₀ = 0.001). Samples were taken 3 d after inoculation to determine the growth of the bacterial pathogen. The means and ses were calculated from triplicate determinations. The experiment was repeated three times with similar results.

Figure 6

NPR1 dependency of AtWRKY18-potentiated defense response. A, Total RNA was isolated from wild-type plants (Wt) with (+) or without (−) SA treatment (2 mm for 3 h) and untreated F₂ progeny of npr1-3 independent transformant lines harboring the 35S-W18Δ construct, separated on a 1.2% (w/v) agarose-formaldehyde gel, and probed with an AtWRKY18 cDNA fragment. The ethidium bromide stain of rRNA is shown for each lane to allow assessment of equal loading. B, Total RNA was isolated from 5.5-week-old wild-type (Wt), npr1-3 mutant with (+) or without (−) SA treatment (2 mm for 3 h), and untreated T₂ progeny of npr1-3 independent transformant lines harboring the 35-W18Δ construct, separated on a 1.2% (w/v) agarose-formaldehyde gel, and probed with a PR-1 fragment. The ethidium bromide stain of rRNA is shown for each lane to allow assessment of equal loading. C, Wild type (Wt), npr1-3 mutant, and T₂ progeny from eight npr1-3 independent transformant lines harboring the 35S-W18Δ construct were inoculated with P. syringae DC3000 (OD₆₀₀ = 0.001) 5.5 weeks after germination. Samples were taken 3 d after inoculation to determine the growth of the bacterial pathogen. The means and ses were calculated from triplicate determinations. The experiment was repeated three times with similar results.

Figure 7

Recognition of W box elements in the AtWRKY18 gene promoter by WRKY DNA-binding proteins. A, Sequences of the PW18 probe (the −1301 to −1330 region of the AtWRKY18 gene promoter) and the mPW18 probe with the TTGAC repeats mutated into TTGAA repeats. B, Sequence-specific binding of PW18 by the AtWRKY18 protein (lane 2). Change of the TTGAC repeats to TTGAA repeats in the mPW18 probe abolished the intensities of retarded bands (lane 4). No retarded bands were detected in the absence of proteins (lanes 1 and 3). C, The PW18 probe recognized DNA-binding activities present at low levels in untreated plants (lane 1) and induced in SA-treated plants (lane 2). The mPW18 probe failed to recognize these DNA-binding activities from untreated (lane 3) or SA-treated plants (lane 4).

Figure 8

Deletion analysis of the AtWRKY18 gene promoter. A, Fusion constructs of different lengths of the AtWRKY18 gene promoter and the GUS reporter gene. Lengths of promoter deletion fragments are indicated from 5′ ends to the translation initiation site. The GUS reporter gene with no upstream promoter sequence (pCH450) was included as a negative control. B, GUS activities in 4-week-old T₂ progeny from 10 independent T₁ transgenic Arabidopsis lines harboring the above promoter deletion constructs 24 h after the treatments. GUS activity on a pool of four T₂ plants from each T₁ line was determined for each treatment and the mean and se were calculated from the 10 independent T₁ lines.

Figure 9

Functional analysis of W box elements in the AtWRKY18 gene promoter. A, Constructs of pCH49 (the +1 to −1,700 promoter sequence of the AtWRKY18 gene fused with the GUS reporter gene) and pCH49 m (the +1 to −1,700 promoter sequence with mutated TTGAA repeats fused with the GUS reporter gene). B, GUS activities in 4-week-old T₂ progeny of 10 independent T₁ transgenic Arabidopsis lines harboring the above promoter constructs 24 h after the treatments. GUS activity on a pool of four T₂ plants from each T₁ line was determined for each treatment and the mean and se were calculated from the 10 independent T₁ lines.